Gérard Le Fur

Selective inhibition of sucrose and ethanol intake by SR 141716, an antagonist of central cannabinoid (CB1) receptors.

Abstract SR 141716, a selective central CB1 cannabinoid receptor antagonist, markedly and selectively reduces sucrose feeding and drinking as well as neuropeptide Y-induced sucrose drinking in rats. SR 141716 also decreases ethanol consumption in C57BL/6 mice. In contrast, blockade of CB1 receptors only marginally affects regular chow intake or water drinking. The active doses of SR 141716 (0.3–3 mg/kg) are in the range known to antagonize the characteristic effects induced by cannabinoid receptor agonists. These results suggest for the first time that endogenous cannabinoid systems may modulate the appetitive value of sucrose and ethanol, perhaps by affecting the activity of brain reward systems.

A Selective Inverse Agonist for Central Cannabinoid Receptor Inhibits Mitogen-activated Protein Kinase Activation Stimulated by Insulin or Insulin-like Growth Factor 1 EVIDENCE FOR A NEW MODEL OF RECEPTOR/LIGAND INTERACTIONS

In the present study, we showed that Chinese hamster ovary (CHO) cells transfected with human central cannabinoid receptor (CB1) exhibit high constitutive activity at both levels of mitogen-activated protein kinase (MAPK) and adenylyl cyclase. These activities could be blocked by the CB1-selective ligand, SR 141716A, that functions as an inverse agonist. Moreover, binding studies showed that guanine nucleotides decreased the binding of the agonist CP-55,940, an effect usually observed with agonists, whereas it enhanced the binding of SR 141716A, a property of inverse agonists. Unexpectedly, we found that CB1-mediated effects of SR 141716A included inhibition of MAPK activation by pertussis toxin-sensitive receptor-tyrosine kinase such as insulin or insulin-like growth factor 1 receptors but not by pertussis toxin-insensitive receptor-tyrosine kinase such as the fibroblast growth factor receptor. We also observed similar results when cells were stimulated with Mas-7, a mastoparan analog, that directly activates the Gi protein. Furthermore, SR 141716A inhibited guanosine 5′-0-(thiotriphosphate) uptake induced by CP-55,940 or Mas-7 in CHO-CB1 cell membranes. This indicates that, in addition to the inhibition of autoactivated CB1, SR 141716A can deliver a biological signal that blocks the Gi protein and consequently abrogates most of the Gi-mediated responses. By contrast, SR 141716A had no effect on MAPK activation by insulin or IGF1 in CHO cells lacking CB1 receptors, ruling out the possibility of a direct interaction of SR 141716A with the Gi protein. This supports the notion that the Gi protein may act as a negative intracellular signaling cross-talk molecule. From these original results, which considerably enlarge the biological properties of the inverse agonist, we propose a novel model for receptor/ligand interactions.

The CB1 receptor antagonist rimonabant reverses the diet-induced obesity phenotype through the regulation of lipolysis and energy balance

We investigated the molecular events involved in the long‐lasting reduction of adipose mass by the selective CB1 antagonist, SR141716. Its effects were assessed at the transcriptional level both in white (WAT) and brown (BAT) adipose tissues in a diet‐induced obesity model in mice. Our data clearly indicated that SR141716 reversed the phenotype of obese adipocytes at both macroscopic and genomic levels. First, oral treatment with SR141716 at 10 mg/kg/d for 40 days induced a robust reduction of obesity, as shown by the 50% decrease in adipose mass together with a major restoration of white adipocyte morphology similar to lean animals. Second, we found that the major alterations in gene expression levels induced by obesity in WAT and BAT were mostly reversed in SR141716‐treated obese mice. Importantly, the transcriptional patterns of treated obese mice were similar to those obtained in the CB1 receptor knockout mice fed a high‐fat regimen and which are resistant to obesity, supporting a CB1 receptor‐mediated process. Functional analysis of these modulations indicated that the reduction of adipose mass by the molecule resulted from an enhanced lipolysis through the induction of enzymes of the β‐oxidation and TCA cycle, increased energy expenditure, mainly through futile cycling (calcium and substrate), and a tight regulation of glucose homeostasis. These changes accompanied a significant cellular remodeling and contributed to a reduction of the obesity‐related inflammatory status. In addition to a transient reduction of food consumption, increases of both fatty acid oxidation and energy expenditure induced by the molecule summate leading to a sustained weight loss. Altogether, these data strongly indicate that the endocannabinoid system has a major role in the regulation of energy metabolism.

Primary structure and functional expression of mouse pituitary and human brain corticotrophin releasing factor receptors

Corticotrophin‐releasing factor (CRF) is the principal hypothalamic factor governing the pituitary‐adrenal axis, but the wide extra‐pituitary distribution of CRF and its receptors suggest a major role for this neuropeptide in the integration of the overall physiological and behavioral responses of an organism to stress. We have cloned a CRF receptor complementary DNA (cDNA) by expression in COS‐7 cells of a cDNA library from the AtT20 mouse pituitary tumour cell line. The cloned mouse cDNA was then used as a probe to isolate a human CRF receptor cDNA from a human brain cDNA library. The mouse and human cDNAs both encode 415 amino acid proteins that are 97% identical, containing seven putative transmembrane domains characteristic of G protein‐coupled receptors. The CRF receptor shows homology with the receptors for growth hormone‐releasing factor, vasoactive intestinal peptide, secretin, parathyroid hormone, and calcitonin. COS‐7 cells transfected with the mouse CRF receptor cDNA bind radiolabelled ovine CRF with high affinity and respond specifically to CRF by accumulation of intracellular cAMP. A 2.7 kb mRNA coding for the CRF receptor could be detected in AtT20 cells and human cortex tissue. PCR analysis also detected the receptor transcript in human pituitary, brainstem, and testis.

Rimonabant reduces obesity-associated hepatic steatosis and features of metabolic syndrome in obese zucker fa/fa rats

This study investigated the effects of rimonabant (SR141716), an antagonist of the cannabinoid receptor type 1 (CB1), on obesity‐associated hepatic steatosis and related features of metabolic syndrome: inflammation (elevated plasma levels of tumor necrosis factor alpha [TNFα]), dyslipidemia, and reduced plasma levels of adiponectin. We report that oral treatment of obese (fa/fa) rats with rimonabant (30 mg/kg) daily for 8 weeks abolished hepatic steatosis. This treatment reduced hepatomegaly, reduced elevation of plasma levels of enzyme markers of hepatic damage (alanine aminotransferase, gamma glutamyltransferase, and alkaline phosphatase) and decreased the high level of local hepatic TNFα currently associated with steatohepatitis. In parallel, treatment of obese (fa/fa) rats with rimonabant reduced the high plasma level of the proinflammatory cytokine TNFα and increased the reduced plasma level of the anti‐inflammatory hormone adiponectin. Finally, rimonabant treatment also improved dyslipidemia by both decreasing plasma levels of triglycerides, free fatty acids, and total cholesterol and increasing the HDLc/LDLc ratio. All the effects of rimonabant found in this study were not or only slightly observed in pair‐fed obese animals, highlighting the additional beneficial effects of treatment with rimonabant compared to diet. These results demonstrate that rimonabant plays a hepatoprotective role and suggest that this CB1 receptor antagonist potentially has clinical applications in the treatment of obesity‐associated liver diseases and related features of metabolic syndrome. (HEPATOLOGY 2007.)

Molecular cloning of a levocabastine-sensitive neurotensin binding site

A search for sequences homologous to the neurotensin receptor cDNA in a rat hypothalamic library has identified a novel neurotensin receptor (NTR‐2). The 1539 bp cDNA encodes a 416 amino acid protein and shows highest homology to the previously cloned neurotensin receptor (NTR‐1) (64% homology and 43% identity). Binding and pharmacological studies demonstrate that NTR‐2 expressed in COS cells recognizes neurotensin (NT) with high affinity as well as several other agonists and antagonists. However, a fundamental difference was found; unlike NTR‐1, NTR‐2 recognizes, with high affinity, levocabastine, a histamine H1 receptor antagonist previously shown to compete with NT for low‐affinity binding sites in brain.

Inhibition of long-term potentiation in rat hippocampal slices by anandamide and WIN55212-2 : reversal by SR141716 A, a selective antagonist of CB1 cannabinoid receptors

It has been reported previously that Δ9-tetrahydrocannabinol and the synthetic cannabinoid agonist HU-210 [(−)-11-OH-Δ8-dimethylheptyl tetrahydrocannabinol] prevent long-term potentiation (LTP) induction in rat hippocampal slices. In this study we confirm that both WIN55212-2 {R-(+)-(2,3-dihydro-5-methyl-3-[{4-morpholinyl} methyl] pyrol [1,2,3-de]-1,4-benzoxazin-6-yl) (1-naphtalenyl) methanone monomethanesulphonate} (3 and 10 μM), another synthetic cannabinoid agonist, and anandamide (10 μM), considered to be the endogenous ligand of cannabinoid receptors, inhibit LTP formation in the Schaffer collateral-CA1 field complex. In addition, we show that SRt417l6A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-lH-pyrazole-3-carboxamide hydrochloride] at 0.1–10 μM, a potent and selective antagonist of CB1 cannabinoid receptors, concentration-dependently reversed the inhibition of LTP induced by both WIN55212-2 and anandamide. These data indicate that cannabinoid receptor agonists inhibit hippocampal LTP formation through CB1 receptor activation and that anandamide could be a candidate for an endogenous neuromessenger involved in memory processes.

Molecular cloning, expression and function of the murine CB2 peripheral cannabinoid receptor.

We have cloned the peripheral cannabinoid receptor, mCB2, from a mouse splenocyte cDNA library. The 3.7 kb sequence contains an open reading frame encoding a protein of 347 residues sharing 82% overall identity with the only other known peripheral receptor, human CB2 (hCB2) and shorter than hCB2 by 13 amino acids at the carboxyl terminus. Binding experiments with membranes from COS-3 cells transiently expressing mCB2 showed that the synthetic cannabinoid WIN 55212-2 had a 6-fold lower affinity for mCB2 than for hCB2, whereas both receptors showed similar affinities for the agonists CP 55,940, delta(9)-THC and anandamide and almost no affinity for the central receptor- (CB1) specific antagonist SR 141716A. Both hCB2 and mCB2 mediate agonist-stimulated inhibition of forskolin-induced cAMP production in CHO cell lines permanently expressing the receptors. SR 141716A failed to antagonize this activity in either cell line, confirming its specificity for CB1.

Hypersensitization of the Orexin 1 receptor by the CB1 receptor: evidence for cross-talk blocked by the specific CB1 antagonist, SR141716.

In the present study, we observed evidence of cross-talk between the cannabinoid receptor CB1 and the orexin 1 receptor (OX1R) using a heterologous system. When the two receptors are co-expressed, we observed a major CB1-dependent enhancement of the orexin A potency to activate the mitogen-activated protein kinase pathway; dose-responses curves indicated a 100-fold increase in the potency of orexin-mediated mitogen-activated protein kinase activation. This effect required a functional CB1 receptor as evidenced by the blockade of the orexin response by the specific CB1 antagonist, N-(piperidino-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-pyrazole-3-carboxamide (SR141716), but also by pertussis toxin, suggesting that this potentiation is Gi-mediated. In contrast to OX1R, the potency of direct activation of CB1 was not affected by co-expression with OX1R. In addition, electron microscopy experiments revealed that CB1 and OX1R are closely apposed at the plasma membrane level; they are close enough to form hetero-oligomers. Altogether, for the first time our data provide evidence that CB1 is able to potentiate an orexigenic receptor. Considering the antiobesity effect of SR141716, these results open new avenues to understand the mechanism by which the molecule may prevent weight gain through functional interaction between CB1 and other receptors involved in the control of appetite.

In vitro functional evidence of neuronal cannabinoid CB1 receptors in human ileum

We investigated the effect of the cannabinoid agonist (+)WIN‐55212‐2 on human ileum longitudinal smooth muscle preparations, either electrically stimulated or contracted by carbachol. Electrical field stimulation mostly activated cholinergic neurons, since atropine and tetrodotoxin (TTX), alone or co‐incubated, reduced twitch responses to a similar degree (85%). (+)WIN‐55212‐2 concentration‐dependently inhibited twitch responses (IC50 73 nm), but had no additive effect with atropine or TTX. The cannabinoid CB1 receptor antagonist SR 141716 (pA2 8.2), but not the CB2 receptor antagonist, SR 144528, competitively antagonized twitch inhibition by (+)WIN‐55212‐2. Atropine but not (+)WIN‐55212‐2 or TTX prevented carbachol‐induced tonic contraction.  These results provide functional evidence of the existence of prejunctional cannabinoid CB1‐receptors in the human ileum longitudinal smooth muscle. Agonist activation of these receptors prevents responses to electrical field stimulation, presumably by inhibiting acetylcholine release. SR 141716 is a potent and competitive antagonist of cannabinoid CB1 receptors naturally expressed in the human gut.